The present invention is directed to a plant-colonizing microorganism, which contains as a chromosomal insert, heterologous DNA coding for a high molecular weight protein having insecticidal activity. The invention is further directed to insecticidal compositions containing such plant-colonizing microorganisms as the active insecticidal agent as well as to the use of such microorganisms in a method of combatting insect pests.
Bacillus thuringiensis (B.t.) subsp. kurstaki is a spore forming soil bacterium which is known for its ability to produce a parasporal crystal which is lethal to a wide subspecies of insect larvae. The crystals, which account for 20-30% of the dry weight of sporulated cultures, are composed primarily of a single, high molecular weight protein (134,000 daltons) which is synthesized only during sporulation.
Whiteley et al (1) reported the isolation of plasmid DNA from Bacillus thuringiensis subsp. kurstaki HD-1, insertion of said DNA into the cloning vector pBR322 and transformation into Escherichia coli strain HB101. Colonies presumed to contain recombinant plasmids were screened for production of an antigen that would react with an antibody made against B.t. crystal protein toxin. One recombinant strain, identified as ES12, was isolated which synthesized a polypeptide of 130,000 daltons which reacted with antibody directed to the crystal protein. Protein extracts of ES12 were toxic to larvae of the tobacco hornworm, Manduca sexta. The amounts of polypeptide produced were very low compared to that produced by B. thuringiensis. This appears to be due to the different methods of regulation of protein production in B. thuringiensis and E. coli.
Klier et al (2) reported that the crystal protein gene of Bacillus thuringiensis subsp. berliner 1715 occurred on both a large host plasmid and on the chromosomal DNA. A DNA sequence corresponding to the chromosomal sequence was inserted into plasmid pBT 15-88 . The inserted sequence of pBT 15-88 was not expressed in E. coli. A 14 Kb BamHI DNA fragment from the 42 megadalton host plasmid was cloned into the BamHI site of pHV33 and this vector was inserted into E. coli Extracts of E. coli containing the recombinant plasmid were immunologically cross-reactive against antibodies directed against purified crystal protein. The polypeptide synthesized by E. coli containing the recombinant plasmid had approximately 10% the activity of that synthesized by sporulating cells of B. thuringiensis. Five-fold concentrated extract of E. coli harboring the recombinant plasmid when spread on cabbage leaves and fed ad libitum were toxic to the larvae of Pierris brassica. Klier et al also inserted pHV33 containing the 14 Kb insert into B. subtilis. The crystal gene was not expressed in vegetative cells; it was expressed in sporulating cells although the amount of crystal protein produced by the sporulating cells was about 10% of that produced by sporulating B. thuringiensis.
Held et al (3) containing DNA fragments of B. thuringiensis subsp. kurstaki by EcoRI digestion and cloned these fragments into the vector Charon 4A. E. coli was infected with a recombinant bacteriophage, C4R6C, consisting of cloning vector Charon 4A and DNA from B. thuringiensis. These infected cells produced protein antigen which was the same size as the B. thuringiensis protoxin and protein extracts were toxic to neonate larvae of Manduca sexta. Hybridization of C4K6C DNA to B. thuringiensis plasmids indicated that the original Charon 4A clone contained the genes of chromosomal, no plasmid origin.
Wong et al (4) reported the nucleotide sequence of the promoter region and part of the coding region of the crystal protein gene from B. thuringiensis subsp. kurstaki HD-1-Dipel. A potential ribosome binding sit of 11 nucleotides was located three nucleotides upstream form the initiator ATG codon. The deduced sequence from the first 333 amino acids of the crystal protein was reported.
U.S. Pat. No. 4,448,885 describes plasmids capable of replicating in an E. coli bacterial host species which contains expressible heterologous DNA coding for a polypeptide of 130,000 daltons which has the immunological properties of the crystal protein of B. thuringiensis. Also disclosed is an E. coli bacterial strain transformed to express a polypeptide of 130,000 daltons which reportedly has immunological properties of the crystal protein of B. thuringiensis. A method of using said bacterial strains to produce an insecticidal effect is also disclosed.
Commercial insecticidal preparations containing spores and crystalline protein produced by Bacillus thuringiensis subsp. kurstaki are available as wettable powders and aqueous suspensions under such names as Dipel.RTM. and Thuricide.RTM.. These materials are used for the control of lepidoptera larvae such as Spruce budworm, cabbage looper, imported cabbage worm, gypsy moth, etc., which prey upon tobacco, cotton, soybeans, etc.
Major limitations to the use of commercial preparations of crystal protein toxin of Bacillus thuringiensis subsp. kurstaki include the need for repeated applications of the insecticidal preparations and limitation of the host range. Another drawback to the use of B. thuringiensis to produce insecticidal crystal protein is that the crystal protein is only produced during the sporulation stage of the B. thuringiensis life cycle. Such a growth phase limitation particularly in an industrial process, can result in inconvenience and excessive time requirements during manufacture. Pressures resulting from growth phase limitations or other factors may result in strains of B. thuringiensis losing their ability to produce the crystals; such acrystalliferous strains do not have insecticidal activity.
Although the isolation of DNA from B. thuringiensis subsp. kurstaki coding for the crystal protein toxin and the insertion of this DNA into expression vectors for the transformation of E. coli or B. subtilis is known, the prior art does not teach that such DNA can be inserted into a plant-colonizing microorganism in the first instance, nor the insertion of this DNA into the chromosome of a plant-colonizing microorganism to produce a plant-colonizing microorganism having insecticidal activity against insect pests. There is no teaching in the art that such plant-colonizing microorganisms can live and grow in the "plant environment" and give contact or systemic season-long insect control avoiding the need for repeated applications of the insecticidal crystal protein.
The delivery of insecticidal protein via a genetically engineered plant-colonizing microorganism which colonizes the "plant environment" and which expresses the insecticidal protein in the plant environment, i.e., on the leaf, stem, stalk, floral parts or root surface is unique and unexpected in view of the prior art which is directed to the production of the insecticidal crystal protein in culture, recovery of the protein from culture and the insecticidal application of preparations of mixtures of crystal protein and spores.